1. Field of the Invention
The present invention relates to a scanning probe microscope and, more preferably, to a scanning probe microscope that is suitable for the observation of the molecular-level reaction of a biological molecule, i.e., a morphological or structural change. The present invention also relates to a molecular structure change observation method using the scanning probe microscope.
2. Description of the Related Art
A scanning probe microscope (SPM) is a generic name for devices which obtain information on a sample by mechanically scanning a probe, and includes, for example, a scanning tunneling microscope (STM), an atomic force microscope (AFM), a scanning magnetic force microscope (MFM), a scanning capacitance microscope (SCaM), a scanning near-field optical microscope (SNOM), and a scanning thermal microscope (SThM).
Recently a nanoindentator, designed to check hardness and other property of a sample by pressing a diamond probe against the sample surface to make an indentation and analyzing how the indentation is formed, has been regarded as one of SPMs, and SPM has become widely used with the above various kinds of microscopic method.
SPM can obtain surface information on a desired sample region through a probe by relatively raster-scanning the probe and the sample in X and Y directions to map and display the information on a monitor TV.
Among SPMs, AFM, in particular, is most widely used. AFM comprises a cantilever having a probe at its free end, an optical displacement sensor which detects the displacement of the cantilever, and a scanning mechanism which relatively scans the probe and the sample. As this optical displacement sensor, an optical lever type optical displacement sensor having high displacement detection sensitivity with a simple arrangement is most widely used.
In a commercially available atomic force microscope, the optical lever type optical displacement sensor applies a light beam having a diameter of 10 to 30 μm to the cantilever and detects a change in the reflection direction of the reflected light in accordance with the warpage of the lever through a two- or four-segments photodetector or the like, thereby outputting an electrical signal reflecting the movement of the probe at the free end of the cantilever.
The atomic force microscope maps the configuration of a sample surface by causing the scanning mechanism to control the Z-direction position of the probe with respect to the sample so as to keep the output of the optical displacement sensor constant while scanning the probe in the X and Y directions with respect to the sample, and displays the resultant information on the monitor of a computer.
The atomic force microscope has attracted attention as a device having the potential of allowing the observation of the manner of movement of a living biological sample in liquid with higher resolution than an optical microscope.
An optical microscope is a device which allows the observation of the manner of movement of a living sample. With the optical microscope, however, a sample cannot be observed with a resolution equal to or less than the wavelength of light due to the diffraction limit.
An electron microscope is a device which can realize high resolution on the nanometer order. The electron microscope, however, does not allow a measurement target to be placed in liquid, and hence does not allow the observation of a living biological sample in liquid.
In contrast to this, the atomic force microscope can be expected to realize high resolution on the nanometer order, and allows even the observation of a sample in liquid. Furthermore, the atomic force microscope can be easily combined with an optical microscope. This is one of the reasons why this device has attracted attention.
An optical microscopic observation method using a caged compound is also known. This observation method allows the observation of a specific reaction of a sample in real time by properly performing uncaging while optically observing the sample in the presence of the caged compound.
A caged compound is a generic term for molecules that are trapped in a “cage” and allowed to be released from the “cage” by an external stimulus. As a caged compound, a combination of a molecule A having physiological activation or fluorescent emission characteristics and a molecule B having photosensitivity is often used, in which the molecules A and B are bonded each other to restrain the physiological activation or fluorescent emission ability of the molecule A in advance, and the bond between the molecules A and B is allowed to be broken by an optical stimulus to release the molecule.
In the optical microscopic observation method using a caged compound, while a sample in liquid containing such a caged compound is optically observed, an optical stimulus is given to the caged compound by applying cage-photolytic light such as ultraviolet light to uncage the caged compound. This makes it possible to observe a reaction between the molecule released by cage release and the sample as an observation target such as a biological molecule. That is, this allows the time-series observation of the state of the sample before, during, and after the reaction between the sample and the molecule released by cage release.
When cage-photolytic light is to be applied, the position where a substance that can react with a sample, the amount of such substance, the timing when the substance is generated, and the like can be arbitrarily changed by controlling the range, strength, time, and the like of cage-photolytic light to be applied. After the cage-photolytic light is applied, the reaction of the sample is tracked by using a fluorescence label. In the optical microscope designed to perform this analysis, light for releasing a protecting group may be caused to come into focus with the target position of a sample being positioned in the visual field. Therefore, cage-photolytic light is applied to a target region upon confirmation of the application position of cage-photolytic light.
The atomic force microscope has the potential of being able to capture the morphological change of one biological molecule in liquid with a resolution on the nanometer order, if the scanning speed is sufficiently increased. In addition, mixing, in liquid, a molecule that reacts with a living molecule such as a protein or nucleic acid makes it possible to directly observe how the living molecule such as a protein or nucleic acid changes its structural form along with a biochemical reaction.
In order to observe the dynamic behavior of a living molecule, i.e., a sample, the sample must not be firmly fixed on a substrate and is partially fixed. Alternatively, the sample is bonded to a molecule fixed to the substrate to observe the dynamic behavior of the sample.
The sample, however, constantly undergoes Brownian motion, even if it is partially fixed to the substrate. That is, the sample is always in a dynamic state regardless of a reaction. It is therefore necessary to discriminate and recognize a change in dynamic state due to Brownian motion and a change in structural form due to a reaction. In practice, however, it is difficult to discriminate and recognize them. It is also difficult to prove that they are really discriminated.
The optical microscopic observation method using a caged compound can clarify the difference between the state before a reaction and the state after the reaction because the timing of releasing a caged compound can be controlled by the application of ultraviolet light. However, the optical microscope is insufficient in spatial resolution to image the structure or form of one molecule, and hence cannot image a change in the structure or form of one molecule.
Note that at the timing of cage release, the force acting between a sample and the probe at the cantilever sometimes changes due to the influence of the radiation pressure of photolytic light. In this case, the force exerted on the sample may reach the level at which the sample is destroyed.
Alternatively, around the cantilever, a solution may fluctuate in refractive index due to the migration caused by the spatial movement of a substance in the solution, e.g., a metal ion, polyion, or protein. As a result, noise that disturbs probe control may be superimposed on a cantilever displacement detection signal. In some case, the disturbance of probe control may reach the level at which the sample or probe is destroyed.